1. Area of the Art
The invention relates generally to BRCA1 detection, and specifically to methods of detecting BRCA1 mutations.
2. Description of the Prior Art
Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
Breast cancer is one of the most commonly diagnosed diseases that affect women. Five to ten percent of breast cancer is associated with inherited factors. Early detection of breast cancer is critical in the medical management of the disease, since treatment of breast cancer at later stages is often futile and disfiguring. Ovarian cancer, although less frequent than breast cancer, is often rapidly fatal and is the fourth most common cause of cancer mortality in American women. Approximately fifty percent of familial breast cancer and eighty percent of familial breast plus ovarian cancer are associated with mutations in the BRCA1 gene located on human chromosome 17q21.3. (1, 2).
BRCA1 appears to be a classical tumor suppressor gene, as BRCA1 protein inhibits the growth of breast and ovarian cancer cell lines and reduces the tumorigenicity of MCF-7 cells. BRCA1 is mutated in some hereditary breast and ovarian cancer. It has been discovered that individuals with the wild-type BRCA1 gene do not have cancer that results from the BRCA1 allele. However, mutations that interfere with the function of the BRCA1 protein are involved in the pathogenesis of cancer. Thus, the presence of an altered (or a mutant) BRCA1 gene which produces a protein having a loss of function, or altered function, directly correlates to an increased risk of cancer.
It has been observed that mutations in the BRCA1 locus in the germline are indicative of a predisposition to breast cancer and ovarian cancer. In addition, somatic mutations in the BRCA1 locus are also associated with breast cancer, ovarian cancer and other cancers, which represent an indicator of these cancers or of the prognosis of these cancers.
The mutational events of the BRCA1 locus can involve deletions, insertions, and point mutations within the coding sequence and the non-coding sequence. Deletions may be the entire gene or only a portion of the gene. Point mutations may result in stop codons, frameshift mutations, or amino acid substitutions. Somatic mutations are those which occur only in certain tissues, e.g., in the tumor tissue, and are not inherited in the germline. Germline mutations can be found in any of a body""s tissues or cells and are inherited. If only a single allele is mutated, a predisposition to breast cancer is indicated.
It is believed that BRCA1 predisposing alleles are recessive to wild-type alleles; that is, cells that contain at least one wild-type BRCA1 allele are not cancerous. However, cells that contain one wild-type BRCA1 allele and one predisposing allele may occasionally suffer the loss of the wild-type allele, either by random mutation or by chromosome loss during cell division. All the progeny of such a mutant cell lack the wild-type function of BRCA1 and may develop into tumors. Thus, the predisposing alleles of BRCA1 are susceptible to cancer, and the susceptibility is inherited in a dominant fashion.
Therefore, the finding of BRCA1 mutations provides both diagnostic and prognostic information. It is believed that many mutations found in tumor tissues will be those leading to the decreased expression of the BRCA1 gene product. However, mutations leading to non-functional gene products would also lead to a cancerous state. The majority of mutant alleles are nonsense (10%) or frameshift (71%) and should produce truncated proteins which are predicted to vary in length from 5% to 99% of the full-length protein. Forty-nine percent of these mutations reside in exon 11 which comprises 61% of the BRCA1 coding region. The full-length BRCA1 cDNA sequence and the coding regions of BRCA1 gene have been described in U.S. Pat. No. 5,747,282 (the ""282 patent), the content of which is incorporated herein by reference.
So far, the assessment of the status of BRCA1 protein in patients has been limited. The testing of mutations in BRCA1 has been primarily focused on genomic sequencing. One system described is the truncation assay system (3). This system relies on PCR to amplify BRCA1 segments from the genomic DNA of patients. These segments are then transcribed and translated in vitro and the protein generated is analyzed by gel electrophoresis. The appearance of protein bands of a size that is smaller than expected indicates the presence of a mutation in that segment of BRCA1. Clearly, this process is laborious and time-consuming. Also, since so many steps are involved, the chances of getting false results may increase.
More recently, there have been reports of protein-based systems for detecting mutations in the BRCA1 gene (Simple Immunoassays for the Detection of BRCA1 in Cells: Journal of Clinical Ligand Assay, Vol. 22, Number 4, 1999 343-347). In this approach, two assays are performed. One measures the presence of full-length protein using one antibody specific for the N-terminal portion of the protein and a second measures the antibody specific for the C-terminal portion of the protein. The second assay uses two antibodies that are both specific for the N-terminal portion of the protein. If a given sample is from an individual who does not have a mutation in the BRCA1 gene, the amount of protein detected by both assays will be comparable. If the sample is from an individual who does have a mutation in the BRCA1 gene and that mutation leads to a truncation of the gene product, then the N-terminal assay measures higher levels of protein present than in the full-length assay. Assays of this type are limited in that they can only detect mutations that result in the generation of truncated protein. Such assays also require the use of two separate measurements which increases the potential for error. A related system is also discussed in U.S. Pat. No. 5,965,377.
Therefore, a need exists for developing a simpler, more effective method for assessing the status of BRCA1 proteins in patients. The finding of BRCA1 mutations provides both diagnostic and prognostic information.
The present invention is based on the discovery that cells with mutated BRCA1 have lower levels of BRCA1 protein compared to cells with wild-type BRCA1. Therefore, by measuring the level of BRCA1 protein contained in a sample of a subject, one may determine the mutational status of BRCA1 in the subject.
Accordingly, one aspect of the present invention provides a method of detecting and determining the presence of BRCA1 gene mutation in a sample. The method includes the steps of determining the amount of BRCA1 polypeptide contained in the sample, and correlating the amount of BRCA1 polypeptide to the presence of the BRCA1 gene mutation in the sample, wherein the amount below a predetermined cutoff value is an indication of the presence of BRCA1 gene mutation in the sample.
Another aspect of the present invention provides a method for determining the presence of a condition associated with BRCA1 mutation comprising the steps of:
(a) determining the amount of BRCA1 polypeptide contained in a sample of the subject, and
(b) correlating the amount of BRCA1 polypeptide to the presence of the condition, wherein the amount below a predetermined cutoff value is an indication of the condition.
In accordance with embodiments of the present invention, the condition associated with BRCA1 mutation includes, but is not limited to, a predisposition to breast cancer or ovarian cancer, and the presence or prognosis of breast cancer or ovarian cancer.
The methods of the present invention are well suited for use to provide both diagnostic and prognostic information on diseases associated with mutations of BRCA1. The methods are also well suited for use in connection with immunoassay, image analysis, flow cytometry, or laser scanning cytometry technologies.
The invention is defined in its fullest scope in the appended claims and is described below in its preferred embodiments.